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, 293 (13), 4653-4663

Both Positional and Chemical Variables Control in vitro Proteolytic Cleavage of a Presenilin Ortholog

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Both Positional and Chemical Variables Control in vitro Proteolytic Cleavage of a Presenilin Ortholog

Swe-Htet Naing et al. J Biol Chem.

Abstract

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His-Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala-Thr) and the other to the non-pathogenic Aβ48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate-enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Keywords: Alzheimer disease; amyloid precursor protein (APP); enzyme kinetics; intramembrane proteolysis; mass spectrometry (MS); membrane enzyme; neurodegenerative disease; presenilin; substrate specificity.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Characterization of mIAP mutants. A, superposition of mIAP (PDB code 4HYC, cyan) and presenilin (PDB code 5A63 chain B, magenta) structures using secondary structure matching (72). TM helices are numbered from N to C terminus. Key sequence motifs are highlighted. AS, active site. B, ab initio model of MRSWT obtained by SAXS. Additional analysis presented in SI Fig. S1. C–E, Michaelis–Menten kinetic analysis of mIAP variant cleavage of Ren390FRET. See also Table 1. F, gel assay of mIAP mutants using MRSWT substrate. Negative control without enzyme is indicated by (−). The uncut substrate and cleavage products (indicated by an arrow) are detected via anti-MBP immunoblot. Molecular mass is indicated in kDa. G and H, cleavage profiles of MRS substrate were generated by Glu-C digestion of the N-terminal product, facilitated by a C-terminal Glu within MBP (black triangle). Major cleavage site is His9-Thr10 (red triangle). Representative LC-MS analysis of MRSWT cleavage sites are shown for all mutants tested. The relative abundance of each reporter peptide, compared with total peptide spectral matches of seven independent peptide products formed by proteolytic cleavage, is presented. Error bars, S.D. from LC-MS analytical replicates. All LC-MS data for mIAP variants are presented in Fig. S6 and Table S1. I, LC-MS spectrum of reporter peptide (z = 3) displaying 350% more 18O incorporation than 16O. Left, the extracted ion current chromatogram for relative abundance of peptide (ALKDAQTNSIHPFHLVIH) with 16O incorporation (middle) versus 18O incorporation at the C terminus of the N-terminal product (right) from enzymatic cleavage. Approximately 11.8% of the peak at 681.7058 is part of the ion cluster for the 16O-labeled peptide containing two 13C isotopes.
Figure 2.
Figure 2.
mIAP cleavage of C100 γ-site. A, Michaelis–Menten kinetic analysis of C100FRET cleavage by WT mIAP in DDM and bicelles and by catalytic mutants (D162A, D220A, and DM) in DDM. B, kinetic data for mIAP treated with increasing (ZLL)2ketone. C–G, gel assay using MCS10 and mIAP, visualized by anti-MBP immunoblot. C, time course in DDM. D, time course in bicelles. E, product formation as a function of mIAP concentration. F, inhibition by (ZLL)2ketone. G, mIAP variants D162A, D220A, and DM are not active. For C–G, molecular mass is indicated in kDa, and negative control without enzyme is indicated by (−). The uncut substrate and cleavage product (indicated by an arrow) are detected as in Fig. 1F. H, LC-MS analysis of MCS10 cleavage sites by mIAP in DDM and bicelles, compared with MRSWT in DDM. Quantification and presentation are as in Fig. 1G. All LC-MS data are presented in Fig. S7 and Table S2. Error bars, S.D.
Figure 3.
Figure 3.
mIAP cleavage of C100 ϵ-site. A, overlay of substrates used in this study and relationship to C100 γ- and ϵ-cleavage sites of γ-secretase. B, mIAP gel assay using MCSTV and MCSGG substrates, visualized by anti-MBP immunoblot. Molecular mass is indicated in kDa. C and D, LC-MS analysis of MCS10, MCSGG, and MCSTV cleavage sites by mIAP in DDM. Quantification and presentation as in Fig. 1G. All LC-MS data are presented in SI Fig. S7 and Table S2. Error bars, S.D.
Figure 4.
Figure 4.
Probing substrate specificity of mIAP using MRS Thr-scanning mutants. A, sequences generated for this study. B, gel assay using WT mIAP and MRS substrate variants, visualized by anti-MBP immunoblot. Molecular mass is indicated in kDa. C–E, corresponding LC-MS analysis of reactions from B with quantification and presentation as in Fig. 1G. All LC-MS data for MRS substrate variants are presented in Fig. S8 and Table S2. Error bars, S.D.

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